The long-term goal of this research project is to understand the molecular mechanisms that control the embryonic formation of glia, and how glia migrate, differentiate, and ensheath axons. The Drosophila CNS midline glia comprise a well-characterized group of glia that ensheath commissural axons and act as an important source of signals that pattern the developing embryo. They constitute an excellent experimental system for studying the molecular genetics of glial development and function, due to the ability to follow glial development and glial:axonal interactions at the single-cell level. In addition, over 50 genes have been identified that are expressed in midline glia that may play important biological roles. To better understand midline glial development, RNA sequencing will be employed to determine the complete midline glial transcriptome, and assess how it changes during development. This will provide a foundation for ultimately understanding the regulatory circuitry that drives midline glial development. The Drosophila midline glia consist of two functionally distinct discrete populations, the anterior midline glia (AMG) and posterior midline glia (PMG). Two key regulators of these alternative midline glial cell fates are the engrailed and runt genes. The transcriptional profile of embryos misexpressing engrailed and runt will be determined to comprehensively understand the molecular differences between AMG and PMG, and discover how engrailed and runt direct these transcriptional programs. The formation and differentiation of midline neurons will be examined in runt misexpression embryos, which are deficient for PMG, to assess whether PMG influence midline neuron development. Notch signaling drives midline glial formation, while the single-minded gene controls midline glial transcription during differentiation. To understand how these important regulators control midline glial development, genetic experiments will be carried-out: (1) to test whether Notch controls single-minded transcription, and (2) to systematically identify the transcriptional targets of single-minded. Additional genes important in midline glial migration, axonal ensheathment, and apoptosis will be identified and studied by screening midline glial-expressed genes by high-resolution confocal microscopy on mutant and transgenic RNAi embryos. Genes encoding transcription factors will be isolated en masse by mutant embryo sorting, and assayed for their affects on 50 midline glial-expressed genes. This will provide a detailed view of how regulatory proteins function combinatorially to regulate functionally-related batteries of genes. Overall, the proposed research will provide a comprehensive molecular description of how midline glia form, differentiate, and interact with axons. This will afford insight into the origins of human neuronal diversity, and how alterations in gene expression during development can lead to human nervous system disorders. PUBLIC HEALTH RELEVANCE: Glia are a major component of the nervous system and are essential for its proper formation and function. Our long-term research goal is to provide a comprehensive molecular description of the factors controlling glial development and function. This project will lead to greater insights into the developmental origins of nervous system disease and possible therapies.